Thermostable FGF10 Polypeptide or Fragment Thereof and Use Thereof

The present invention relates to thermostable engineered Fibroblast Growth Factor 10 (FGF10) or the fragment thereof having improved thermal stability compared to the wild-type FGF10 and the use thereof in cultivation of spheroids and organoids, regenerative medicine, or other related medical applications or cosmetics. The present invention further relates to a culture medium comprising FGF10 suitable for proliferation and differentiation of the human embryonic stem cells as well as formation and differentiation of spheroids, and organoids.

Fibroblast Growth Factor 10 (FGF10, also referred to as keratinocyte growth factor 2, KGF-2, KGF2 or rhKGF2) belongs to one of five paracrine-acting subfamilies of fibroblast growth factors that interact with heparin. Together with closely related FGF3, FGF7 and FGF22, FGF10 forms so-called FGF7 subfamily that is unique when compared to other FGFs because its members are secreted exclusively by the mesenchyme, and specifically activate the “b” isoforms of receptor FGFR1 (FGFR1b) and FGFR2 (FGFR2b) present in the overlying epithelium (Itoh, N. & Ornitz, D. M., 200420:563-569). FGF10 plays an important role in organogenesis and tissue patterning in the embryo, and mediates wound healing and tissue homeostasis in adult mammals via regulation of cell proliferation and cell differentiation (Beenken, A. & Mohammadi, M., 20098:235-253). FGF10 has been shown to mediate epithelial-mesenchymal interactions, which are essential to lung development and regeneration after various stresses (Tong, L. et al., 20166:21642). FGF10 is further required for development of limb, thyroid, pituitary, kidney, teeth, hair follicles, salivary gland, inner ear, thymus, glandular stomach, and pancreas (Itoh, N., 201628:63-69).

The human FGF10 cDNA encodes a 208 amino acid residue protein with a hydrophobic N-terminal signal peptide of 37 amino acids. FGF10 is highly conserved among the vertebrate species, in both gene structure and amino-acid sequence (Ornitz, D. M & Itoh, N., 20012:3005) Across the vertebrate species, orthologous FGF10 proteins share about 85% amino acid sequence identities as well as similar biological functions (Ornitz, D. M & Itoh, N., 20154:215-266). FGF10 contains the conserved ˜120-amino acid core region (amino acids 75-166 and 176-205) that is homologous (˜30-60%) to other paracrine FGF members (Emoto, H. et al., 1997272:23191-23194) and that adopt a β-trefoil fold consisting of 12 β-strands (Yeh, B. K. et al., 2003100:2266-2271). The core region of FGF10 bears most of the amino acids that are important for interaction with FGFR2 receptor.

The selective interaction of FGF10 with FGFR2b receptor leads to autophosphorylation of the receptor and later to the activation of intracellular signalling pathways (RAS-MAPK, PI3K-AKT, and PLCγ1), that promote cell survival, induce a mitogenic cell response, and mediate cell mobility. Thanks to its ability to regulate epithelial proliferation, lineage commitment during embryonic development and post-natal life, and ability to stimulate epithelial regeneration after injury demonstrated on rodent animal models, the main therapeutical potential FGF10 lies in preventing alveolar cell injuries and restoring the normal epithelial function in damaged lungs (Yuan, T. et al., 20189:418). The other potential therapeutical applications of FGF10 includes wound healing (U.S. Pat. No. 6,653,284 B2), treatment of heart diseases (Rochairs, F. et al., 2014104:432-442), or treatment of obesity and metabolic diseases (Fisher, C. et al., 20178:2079). Besides the therapeutic applications, FGF10 is often used as a component of cultivation media for various embryonic stem cells, spheroids and organoids (U.S. Pat. No. 6,692,961 B1; Rabata, A, et al., 202021:574).

Practical potential of FGF10 is, however, limited by its intrinsic instability demonstrated by its short half-life (lower than 24 hours in cell-free medium), low thermostability, and vulnerability to proteolytic degradation connected with rapid loss of its biological activity (Buchtova, M. et al., 201572:2445-2459; Chen, G. et al., 201230:623-630). The low stability of FGF10 represents a significant obstacle for storage, transport, and practical use of this protein. Due to its low stability, FGF10 must be replaced frequently in cell cultures and the fluctuations of concentration may negatively influence cell fitness. The same goes for therapeutic applications as unfolded or aggregated protein forms arising as a result of the degradation may be toxic or immunogenic (Chen, B. L. & Arakawa, T., 199685:419-426).

A typical process of FGF10 stabilization is represented by addition of heparin. Heparin binding protects FGF10 from heat and acid denaturation and prolongs its half-life (Buchtova, M. et al., 201572:2445-2459). Nevertheless, heparin use is not physiological as it is produced exclusively by mast cells and its anticoagulation properties can further influence the fragile balance in the cellular environment. To use FGF10 in medical and cosmetic applications, different ways of obtaining higher stability must be developed.

Other strategies have been adopted to stabilize FGF10 without the use of heparin. The one described in WO 2013/082196 (or U.S. Pat. No. 10,039,807) uses heparin mimicking sulfonate polymers and co-polymers in conjunction with FGF10 to achieve stability. Others use reducing agents (U.S. Pat. No. 7,754,686), sucrose octa sulphate addition (U.S. Pat. No. 5,202,311), hydroxypropyl cellulose addition (U.S. Pat. No. 5,189,148), glucan sulphate addition (EP 0345660), polyanion-PEG conjugates (Khondee, S., et al., 201112:3880-3894), or chelating agents (U.S. Pat. No. 5,217,954) to stabilise FGF polypeptides. Patent document U.S. Pat. No. 8,304,387 describes novel stable formulation of keratinocyte growth factor (KGF) useful for lyophilisation, where KGF is used in complex with sugars, surfactants, or bulking agents. EP 750628 B1 describes FGF10 produced by recombinant techniques. Such polypeptide, or polynucleotide encoding such polypeptide, can be used for therapeutic purposes, for example, promoting healing of wounds, burns and ulcers, to prevent neuronal damage due to neuronal disorders, and to prevent skin aging and hair loss. However, this form of polypeptide is not stabilized by any means.

An alternative approach to the addition of stabilizing additives is represented by protein engineering. Protein engineering introduces substitutions in the amino acid sequence that enhance the protein stability. Patent document EP 0950100 B1 relates to engineered forms of FGF10 polypeptide truncated at its N-terminal or C-terminal parts, and carrying the C-terminal substitutions R194E, R194Q, K191E, K191Q, R188E, R188Q that were constructed with a goal to enhance level of protein expression and solubility in, and that can be used in therapeutic applications. However, they neither state nor support that the mutations or the truncation would affect thermal stability or half-life of the presented FGF10 variants. Patent document U.S. Pat. No. 6,653,284 B2 relates to liquid and lyophilized formulations of FGF10 polypeptides truncated either at the N-terminal or C-terminal parts for therapeutic use requiring soft issue growth and regeneration. For none of the truncated forms of FGF10, enhanced thermal stability or half-life have been demonstrated. Patent document WO 2001/002433 A1 relates to mutant forms of FGF10 polypeptides truncated at the N-terminal part and carrying the substitutions R68G, R68S, R68A, R78A, R80A, K81A, K87A, K91A, K136A, K137A, K139A, K144A, K148E, K149E, K151A, K153A, K155A, R174A, K183A, K183Q, K183E, R187A, R188A, R188E, or K191E that show enhanced activity, increased stability, higher yield, or better solubility and can be used in therapeutic application to promote wound healing. The other potentially stabilizing substitutions of FGF10 polypeptide, N105D, C106F, K144R, K153M, and I156R have been recently identified by in silico design, however their experimental verification have not been provided (Alizadeh, A. K., et al., 202138:197-209).

The present invention presents isolated thermostable engineered polypeptide that is derived from human FGF10 polypeptide (SEQ ID NO: 1) and can be used in cultivation of spheroids and organoids, regenerative medicine or other related medical applications, or cosmetics.

The drawback resulting from natural instability of FGF10 polypeptide is overcome by the present invention that presents an isolated thermostable engineered polypeptide that possesses FGF10 activity and has 85% sequence identity to a sequence SEQ ID NO: 1 or a functional fragment thereof.

The subject-matter of the present invention is a thermostable FGF10 polypeptide possessing FGF10 activity and having or comprising at least 85% sequence identity of SEQ ID NO:3 that has an amino acid sequence from Ser69 to Ser208 of SEQ ID NO:1, or a fragment thereof, wherein the thermostable FGF10 polypeptide or the fragment thereof comprising at least an amino acid substitution L152F.

According to a prefered embodiment of the present invention the thermostable FGF10 polypeptide has or comprises SEQ ID NO:3, or the fragment thereof, wherein a thermostable FGF10 polypeptide or the fragment thereof comprising at least an amino acid substitution L152F.

Prefered embodiment of the present invention discloses the thermostable FGF10 polypeptide or the fragment thereof further comprising at least one, or at least two, or at least three amino acid substitutions selected from a group consisting of V123I, Q175E, and N181D. This means that the polypeptide according to the invention always exhibits at least L152F substitution; or combination of two, three or four substitutions combining L152F with V123I and/or Q175E and/or N181D.

The amino acid sequence of human FGF10 polypeptide (SEQ ID NO: 1) consists of 208 amino acids, where N-terminal amino acid region MWKWILTHCA SAFPHLPGCC CCCFLLLFLV SSVPVTC (SEQ ID NO: 2) of the amino acid sequence SEQ ID NO: 1 corresponds to an N-terminal signal peptide, that directs the protein translocation and that is usually removed in the mature polypeptide. Signal peptide thus may or may not be present in the amino acid sequence of FGF10 polypeptide according to the present invention. In some embodiments, such as recombinant production of FGF10 polypeptide in, it may be replaced just by single amino acid methionine (M).

The mature human FGF10 polypeptide may be further truncated at its N-terminus up to 31 amino acids without loss of its biological activity and thus consisting of amino acid sequence Ser69-Ser208 with or without N-terminal Met (amino acid numbering corresponds to SEQ ID NO: 1).

It is further known in the art that FGF10 polypeptides of various vertebrate species have about 85% sequence identity, while performing analogous functions and having similar biological activity.

The subject of the present invention relates to an isolated engineered thermostable FGF10 polypeptide exhibiting FGF10 activity and comprising:

Preferably, the length of the thermostable FGF10 polypeptide sequence is up to 228 amino acids, more preferably up to 190 amino acids.

The amino acid marked in bold and underlined in SEQ ID NO: 3 is mutated in the position 84 of this amino acid sequence; which corresponds to the amino acid substitution L152F in the amino acid numbering according to SEQ ID NO: 1.

In some embodiments, the thermostable polypeptide further comprises at least one, or at least two, or at least three of the following amino acid substitutions:

In some embodiments, the thermostable FGF10 polypeptide may carry at the N-terminal part of SEQ ID NO: 3 at least part or all of the amino acid sequence MWKWILTHCA SAFPHLPGCC CCCFLLLFLV SSVPVTCQAL GQDMVSPEAT NSSSSSFSSP SSAGRHVR (SEQ ID NO: 4). In other embodiments, the thermostable FGF10 polypeptide may carry at the N-terminal part of SEQ ID NO: 3 methionine (M). In some embodiments, the thermostable FGF10 polypeptide may carry at its N-terminal or C-terminal parts short fusion amino acid sequences (known as tags) up to a maximum length of 20 amino acids. Said embodiments of the extensions at the N-terminus and C-terminus of the thermostable FGF10 polypeptide sequence can be combined.

According to the other prefered embodiment of the present invention the thermostable FGF10 polypeptide further carries:

In a preferred embodiment, the present invention discloses a thermostable FGF10 polypeptide having or comprising a sequence with at least 85% sequence identity to the sequence SEQ ID NO: 5:

Wherein the amino acid marked in bold and underlined must be retained.

Preferably, the length of the thermostable FGF10 polypeptide sequence is up to 228 amino acids, more preferably up to 190 amino acids.

The amino acid sequence SEQ ID NO: 5 corresponds to the Leu40-Ser208 fragment of SEQ ID NO: 1. The amino acid marked in bold and underlined in SEQ ID NO: 5 is mutated in the position 113 of this amino acid sequence; which corresponds to the amino acid substitution L152F in the numbering according to SEQ ID NO: 1.

According to the other prefered embodiment of the present invention the thermostable FGF10 polypeptide has or comprises at least 85% sequence identity of SEQ ID NO:5 that has an amino acid sequence from Leu40 to Ser208 of SEQ ID NO:1, or the fragment thereof, wherein the thermostable FGF10 polypeptide or the fragment thereof comprises at least an amino acid substitution L152F.

According to the other prefered embodiment of the present invention the thermostable FGF10 polypeptide has or comprises SEQ ID NO:5, or the fragment thereof, wherein a thermostable FGF10 polypeptide or the fragment thereof comprises at least an amino acid substitution L152F.

In some embodiments, the thermostable FGF10 polypeptide further comprises at least one, or at least two, or at least three of the following amino acid substitutions:

According to the other prefered embodiment of the present invention the thermostable FGF10 polypeptide or the fragment thereof further comprises at least one, or at least two, or at least three amino acid substitution selected from a group consisting of V123I, Q175E, and N181D.

In some embodiments, the thermostable polypeptide may carry at the N-terminal part of SEQ ID NO. 5 at least part or all of the amino acid sequence MWKWILTHCA SAFPHLPGCC CCCFLLLFLV SSVPVTCQA (SEQ ID NO: 6). In other embodiments, the thermostable polypeptide may carry at the N-terminal part of SEQ ID NO: 5 methionine (M). In some embodiments, the thermostable polypeptide may carry at its N-terminal or C-terminal parts short fusion amino acid sequences (known as tags) up to a maximum length of 20 amino acids. Said embodiments of the extensions at the N-terminus and C-terminus of the thermostable polypeptide sequence can be combined.

In the detailed description of the invention and in the examples of the invention, the numbering of amino acid substitutions according to the sequence SEQ ID NO: 1 is used, i.e., numbering relative to the entire amino acid sequence of human FGF10 polypeptide, even when truncated amino acid sequences are used.

The thermostable FGF10 polypeptides or the fragments thereof according to the present invention possess unmodified FGF10 activity in 4MBr-5 cell proliferation assay, where lung epithelial 4MBr-5 cells express the FGF receptor 2b (FGFR2b), which is a natural ligand of the FGF10 polypeptide (see). Advantageously subjected thermostable FGF10 polypeptides or the fragments thereof according to the invention show stable and unchanged biological activity at 37° C. for prolonged periods of time (see).

The thermostable FGF10 polypeptides of the present invention are particularly advantageous because they are significantly more stable when compared with FGF10 wild-type or the fragment thereof. This stability is due to the inherent stability of the polypeptide molecule. No other compounds promoting protein stability, such as additives (i.e., heparin) or carrier molecules (i.e., BSA), have to be added. At the same time, the biological activity of thermostable FGF10 polypeptides is preserved. Disclosed thermostable FGF10 polypeptides can be used in research and clinical applications.

The thermostable FGF10 polypeptides or the fragments thereof according to the present invention possess unmodified FGF10 activity and increased melting temperature by 7 to 19° C., preferably by 10 to 19° C. compared to the wild-type FGF10 polypeptide. All thermostable FGF10 polypeptides according to the present invention were constructed by side-directed mutagenesis or using the artificial gene synthesis, subcloned into expression vector pET28b, purified to homogeneity (protein purity ≥95% as determined by SDS-PAGE electrophoresis) and subsequently characterized for stability (melting temperature determination) and biological activity.

More preferred are thermostable FGF10 polypeptides up to 228 amino acids in total length (more preferably up to 190 amino acids in total length) having or comprising at least 85% sequence identity to the amino acid sequences selected from SEQ ID NO: 5, and SEQ ID NO: 7 to SEQ ID NO: 13 or the fragments thereof.

According to the other preferred embodiment of the present invention the thermostable FGF10 polypeptide is selected from a group of the thermostable FGF10 polypeptides comprising SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, or SEQ ID NO:13.

In some embodiments, the thermostable polypeptide of any of the sequences SEQ ID NO: 5, and SEQ ID NO: 7 to SEQ ID NO: 13 may carry at its N-terminal part at least part or all of the amino acid sequence MWKWILTHCA SAFPHLPGCC CCCFLLLFLV SSVPVTCQA (SEQ ID NO: 6). In other embodiments, the thermostable polypeptide of any of the sequences SEQ ID NO: 5, and SEQ ID NO: 7 to SEQ ID NO: 13 may carry at its N-terminal part methionine (M). In some embodiments, the thermostable polypeptide of any of the sequences SEQ ID NO: 5, and SEQ ID NO: 7 to SEQ ID NO: 13 may carry at its N-terminal or C-terminal parts short fusion amino acid sequences (known as tags) up to a maximum length of 20 amino acids. Said embodiments of the extensions at the N-terminus and C-terminus of the thermostable polypeptide sequence can be combined.

According to the other prefered embodiment of the present invention the thermostable FGF10 polypeptide further carries:

Another embodiments of the present invention disclose a thermostable FGF10 polypeptide possesing FGF10 activity wherein the thermostable FGF10 polypeptide according the present invention further carries SEQ ID NO:6 at the N-terminus any of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO: 13.

The biological activity of FGF10 polypeptides, or fragments thereof, or muteins thereof according to the present invention can be quantitatively expressed by median effective dose, ED(concentration of polypeptide needed to achieve half of the maximal biological effect), for the proliferation of 4MBr-5 cells, with an EC50 for this effect <15 ng/ml, preferably <12 ng/mL. The biological activity of FGF10 can be evaluated by a cultured lung epithelial proliferation assay as previously described (Rubin, J. S. et al., 198986:802-806).

More preferred embodiments of the present invention disclose the thermostable FGF10 polypeptide according to the present invention for use in regenerative medicine, preferably for lung epithelial regeneration after various stresses or for curing of wounds and ulcers or other related medical applications, such as cardiac treatment.

More preferred embodiments of the present invention further disclose the use the thermostable FGF10 polypeptide according to the present invention in cosmetics, preferably support of collagen synthesis, skin strength, anti-aging treatment, and non-therapeutic stimulation of hair growth.

More preferred embodiments of the present invention disclose a culture medium for proliferation and differentiation of the human embryonic stem cells, or formation and differentiation of spheroids and organoids, preferably lung organoids, stomach organoids, liver organoids, pancreas organoids, and prostate organoids comprising an effective amount of the thermostable FGF10 polypeptide according to the present invention in the range of 10 ng/ml to 500 ng/ml of culture medium.

These and other features, objects, and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention.

The definition of certain terms as used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.